Natural Product Investigation in Lichens: Extraction and HPLC Analysis of Secondary Compounds in Mycobiont Cultures.

Chromatography techniques facilitate separation, purification, and identification of secondary compounds (natural products) in lichens and their mycobiont cultures. In particular, high-performance liquid chromatography (HPLC) plays a vital role in the identification of lichen substances because of its high sensitivity, speed, and reliability with the minimal sample. Therefore, we describe the extraction and HPLC protocol for the investigation of secondary compounds with a special focus on lichen mycobiont cultures.

Insights into in vitro phenotypic plasticity, growth and secondary metabolites of the mycobiont isolated from the lichen Platygramme caesiopruinosa.

The choice of inoculum for successful isolation and establishment of axenic lichen mycobiont culture is a key step towards eliminating endolichenic and lichenicolous fungi and other microbial contamination. The nutritional requirements of each lichen species are unique. This work reports on the isolation, phenotypic plasticity, growth and secondary metabolites from mycobiont culture of the pantropical lichen Platygramme caesiopruinosa. Media composition [Malt yeast extract (MY), Modified Murashige and Skoog (MMS) and Lilly and Barnett (LB) media] was optimized to determine nutritional requirements for optimal growth of this species as assessed by dry biomass and the occurrence of secondary metabolite. Furthermore, the role of different carbon sources in affecting growth, growth stages, phenotypic plasticity, biomass and spectrum of secondary metabolites produced of this mycobiont was examined. The molecular identity of the mycobiont culture was confirmed by amplifying mitochondrial small subunit (mtSSU) sequences. Cultures showed optimum biomass production in MY medium with 10% sucrose. The secondary metabolite profiles for each culture treatment were characterized using High-performance Thin-Layer Chromatography (HPTLC) and Gas Chromatography with Mass Spectrometric (GC-MS) analysis. The HPTLC spectral comparison, phenolic and iodine confirmatory analysis revealed the absence of phenolic metabolites and the presence of non-phenolic metabolites in mycobiont extracts, while GC-MS analysis revealed the biosynthesis of side chain fatty acids, hydrocarbons and sugar alcohol in mycobiont cultures treated with 10% supplemented sucrose as a carbon source.